Isolation of cellular material under microscopic visualization

ABSTRACT

A method of microdissection which involves forming an image field of cells of the tissue sample utilizing a microscope, identifying at least one zone of cells of interest from the image field of cells which at least one zone of cells of interest includes different types of cells than adjacent zones of cells, and extracting the at least one zone of cells of interest from the tissue sample. The extraction is achieved by contacting the tissue sample with a transfer surface that can be selectively activated so that regions thereof adhere to the zone of cells of interest to be extracted. The transfer surface includes a selectively activatable adhesive layer which provides, for example, chemical or electrostatic adherence to the selected regions of the tissue sample. After the transfer surface is activated, the transfer surface and tissue sample are separated. During separation, the zone of cells of interest remains adhered to the transfer surface and is thus separated from the tissue sample, the zone of cells of interest may then be molecularly analyzed.

[0001] This patent application is related to Provisional PatentApplication Ser. No. 60/036,927, filed Feb. 7, 1997, Patent CooperationTreaty Application Ser. No. PCT/US96/16517, filed Oct. 9, 1996 and nowpending, which is a continuation-in-part of U.S. patent application Ser.No. 08/544,388, filed Oct. 10, 1995 and now pending, which is acontinuation-in-part of Patent Cooperation Treaty Application Ser. No.WO 95/23960 (PCT/US95/02432), filed Mar. 1, 1995 and now pending, whichis continuation of U.S. patent application Ser. No. 08/203,780, filedMar. 1, 1994 and now pending, the disclosures of which are herebyincorporated by reference.

TECHNICAL FIELD

[0002] The present invention relates to methods and devices for themolecular analysis of cellular samples. More particularly, the presentinvention relates to methods and devices for the microdissection andmolecular analysis of cellular samples which may be used in combinationwith a number of different technologies that allow for analysis ofproteins, such as enzymes, and mRNA and DNA from substantially purepopulations or subpopulations of particular cell types.

BACKGROUND ART

[0003] Many diseases are now understood at the molecular and geneticlevel. Analysis of such molecules is important for disease diagnosis andprognosis. Previous methods for direct extraction of cellular tissuematerial from a tissue sample are limited because the extractionreflects only the average content of disease associated markers. Inreality, tissues are very heterogeneous and the most diagnostic portionsof the tissue may be confined to a few hundred cells or less in alesion.

[0004] Normal tissue samples contain a variety of cell types surroundingand adjacent to the pre-invasive and invasive tumor cells. A region ofthe tumor tissue subject to biopsy and diagnosis as small as 1.0 mm cancontain normal epithelium, pre-invasive stages of carcinoma, in-situcarcinoma, invasive carcinoma, and inflammatory areas. Consequently,routine scraping and cutting methods will gather all of these types ofcells, and hence, loss of an allele will be masked by presence of anormal copy of the allele in the contaminating non-malignant cells.Existing methods for cutting away or masking a portion of tissue do nothave the needed resolution. Hence the analysis of genetic results bythose previous methods are always plagued by contaminating alleles fromnormal cells, undesired cells or vascular cells.

[0005] The molecular study of human tumors is currently limited by thetechniques and model systems available for their characterization.Studies to quantitatively or qualitatively assess proteins or nucleicacid expression in human tumor cells are compromised by the diverse cellpopulations present in bulk tumor specimens. Histologic fields ofinvasive tumor typically show a number of cell types including tumorcells, stromal cells, endothelial cells, normal epithelial cells andinflammatory cells. Since the tumor cells are often a relatively smallpercentage of the total cell population it is difficult to interpret thesignificance of net protein or nucleic acid alterations in thesespecimens.

[0006] Studies of human tumor cells in culture do not account for thecomplex interactions of the tumor cells with host cells andextracellular matrix, and how they may regulate tumor cell proteaseproductivity or activation. Immunohistochemical staining allows one toexamine enzyme distribution in regions of tumor invasion, however,results vary with tissue fixation and antibody-antigen affinity, andprovide only a semi-quantitative assessment of protein levels.Furthermore, quantitative interpretation of staining results iscomplicated by the variability of staining patterns within tissuesections, subjective evaluation of staining intensity, and thedifficulty in interpreting the significance of stromal staining. Inaddition, many antibodies utilized in the study of proteases do notdifferentiate pro-enzyme from active enzyme species. Assays of enzyme ormRNA levels from homogenates of human tumors does not account for eitherthe mixed population of cells within the specimens, or the concomitantpathophysiologic processes which may be occur in the tissue.

[0007] Prior methods of study have not allowed investigators tospecifically examine genetic alterations in pre-invasive lesions. Eventhe most sophisticated genetic testing techniques to date have been oflimited value because the input DNA, RNA or proteins to be analyzed arenot derived from pure cell populations exhibiting the diseasemorphology. Several methods have been reported for tissuemicrodissection to address this problem, including gross dissection offrozen tissue blocks to enrich for specific cell populations,irradiation of manually ink stained sections to destroy unwanted geneticmaterial, touch preparations of frozen tissue specimens andmicrodissection with manual tools. These methods, however, are notsufficiently precise and efficient for routine research or highthroughput clinical molecular diagnostic applications. Manualmigrodissection, for example, has good precision but is time consuming,labor intensive, requires a high degree of manual dexterity, and is notgenerally suitable for the ordinary technologist.

[0008] The present inventions provides a novel improved means tospecifically examine genetic alterations in pre-invasive lesions ofcommon epithelial tumors such as breast and prostate carcinoma. Inparticular, the present invention permits the microsampling of as few asone cell, with RNA and DNA extraction of the sampled cell. This methodhas been demonstrated to be extremely sensitive and to surpass previousand current technologies by more than two orders of magnitude. It hasallowed the sensitive detection of loss of heterozygosity in earlypre-invasive lesions being a gateway to the discovery of, for example,new genetic loci on chromosome 11 for breast cancer and a new geneticloci on chromosome 8 for prostate carcinoma.

[0009] The practice of the invention further permits the construction ofgenetic libraries from the extracted material. Thus, libraries frompredetermined cells of interest, particularly abnormal cells, may beconstructed and compared to libraries made from close-by, or adjacent,other cells, such as normal cells. Such libraries may be used, forexample, to compare one or more specific genetic loci, the expression ofone or more RNAs, particularly mRNAs, to isolate and/or clone one ormore specific nucleic acid, and the like.

SUMMARY OF THE INVENTION

[0010] It is accordingly one object of the present invention to providea method of identifying specific cells in cellular tissue sample.

[0011] Another object of the present invention is to provide a method ofdirect extraction of specific cells from a cellular tissue sample.

[0012] It is a further object of the present invention to provide anautomated method of identifying specific cells in cellular tissuesample.

[0013] A further object of the present invention is to provide anautomated method of direct extraction of specific cells from a cellulartissue sample.

[0014] A still further object of the present invention is to provide amethod of obtaining pure cell populations from a cellular tissuesamples.

[0015] According to these and further objects of the present inventionwhich will become apparent as the description thereof proceeds, thepresent invention provides for a method of direct extraction of cellularmaterial from a tissue sample which involves:

[0016] a) providing a slide-mounted tissue sample;

[0017] b) forming an image field of cells of the tissue sample utilizinga microscope;

[0018] c) identifying at least one zone of cells of interest from theimage field of cells, the at least one zone of cells of interestincluding different types of cells than adjacent zones of cells; and

[0019] d) extracting the at least one zone of cells of interest from thetissue sample.

[0020] In another embodiment, the present invention provides a method ofdirect extraction of cellular material from a tissue sample whichinvolves:

[0021] a) providing a tissue sample;

[0022] b) contacting the tissue sample with a selectively activatablesurface which can be activated to provide selective regions thereof withadhesive properties;

[0023] c) identifying at least one portion of the issue sample which isto be extracted;

[0024] d) selectively activating a region of the transfer surface whichcorresponds to and is in contact with the at least one portion of thetissue sample so that the activated region of the transfer surfaceselectively adheres to the at least one portion of the tissue sample;and,

[0025] e) separating the transfer surface from the tissue sample whilemaintaining adhesion between the activated region of the transfersurface and the at least one portion of the tissue sample such that theat least one portion of tissue sample is extracted from the remainingportion of the tissue sample.

[0026] In a preferred embodiment, the activation of the selectivelyactivatable surface is accomplished with a laser.

BRIEF DESCRIPTION OF DRAWINGS

[0027]FIG. 1 is a functional system diagram depicting how a tissuesample is microscopically imaged, displayed on a display monitor, andhow a region of the imaged sample is selected and identified forsubsequent microdissect on and analysis.

[0028]FIG. 2a-2 c are a series of functional system diagrams whichdepict how a zone of tissue sample is extracted from the slide-mountedtissue sample according to one embodiment of the present invention.

[0029]FIG. 3 is a schematic illustration of an alternative device forextracting sample zones from the slide-mounted tissue sample.

[0030]FIGS. 4a and 4 b are schematic diagrams of a manual extractiontool manipulator which can be used together with the extraction deviceof FIG. 3 according to the present invention.

[0031]FIG. 5 is a functional system diagram which shows how a zone ofsample tissue can be directed to an appropriate analysis protocol.

[0032]FIGS. 6a and 6 b show the expression of MMP-2 in ten invasivecolon carcinoma cases (FIG. 6a) and in five cases of invasive breastcarcinoma (FIG. 6b) as compared to normal colonic mucosa from the samepatients.

[0033]FIG. 7 shows SSCP analysis of MMP-2 activation site.

[0034]FIGS. 8a-8 d are schematic illustrations of the sequential stepsof an adhesive transfer method according to one embodiment of thepresent invention.

[0035]FIG. 9 schematically depicts the laser capture microdissectiontechnique.

[0036]FIGS. 10a-10 b schematically depict various embodiments of lasercapture microdissection apparatuses.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0037] The present invention is directed to a method of analyzingcellular material on a molecular or genetic level which involves:visualizing a field of cells in a tissue sample under a microscope,contacting an identified area with a surface which simultaneouslydissolves, extracts and/or retains the cellular material of interest,and transferring the cellular material of interest to a suitableanalysis system. The present invention is particularly applicable to theanalysis of local tissue polypeptides proteins, such as enzymes andantigens, as well as DNA, RNA, particularly mRNA, lipids, carbohydrates,and other biological molecules and assemblies thereof.

[0038] According to one embodiment, the present invention is directed toadhesive transfer methods which involve microscopic visualization andtransfer of cellular material to a procurement or transfer surface.

[0039] The present invention is also directed to a fully automatedsystem whereby a tissue can be visualized, for example, on a screen, sothat a precise field of cells of interest can be identified, forexample, by a variety of labels, histochemical stains, antibodies, etc.,circumscribed or their location otherwise demarcated, and then beextracted and analyzed, either manually or automatically, or by acombination of the two.

[0040]FIG. 1 is a functional system diagram which shows how a tissuesample is microscopically imaged, displayed on a display monitor, andhow a region of the imaged sample is selected and identified forsubsequent microdissection and analysis. As depicted in FIG. 1, a tissuesample 1 is provided on a surface, such as a glass slide 2,formicroscopic examination and imaging. The sample tissue 1 can be fixed onthe glass slide 2 according to any conventional method, includingattachment to the glass slide 2 with an agarose gel, fixing the tissuesample in paraffin, etc.

[0041] The glass slide 2 having the sample tissue 1 mounted thereon isplaced on the stage of a microscope. The microscope, generally indicatedby reference numeral 3, receives an image of the tissue sample 1. Animaging device, such as a video camera, (not shown) is connected to themicroscope 3. The imaging device receives the image of the sample tissue1 from the microscope 3 and displays the image of the tissue sample onan imaging display device, such as display monitor 4.

[0042] The image of the sample tissue 1 is limited to the “field” of themicroscope 3 for any given image. As indicated in FIG. 1, the field ofthe sample tissue image may include several zones, “A”, “B”, “C”, and“D” of different types of cells which can be optically distinguished byutilizing a suitable dye(s), labeled molecules such as antibodies orfragments thereof, to stain or otherwise differentiate the predeterminedcells of interest in the tissue sample. For exemplary purposes, FIGS. 1and 2a-2 c assume that zone “B” is the zone of cellular material ofinterest. The image on the display monitor 4 is used by the operator toselect and identify one or more zones of the tissue sample 1 which areof interest. According to one embodiment of the present invention, afterthe zone(s) of interest are selected and identified, the operatormanually manipulates a device to extract the identified zone(s) from theglass slide 2. The identification of the cells of interest may also bedone automatically through image analysis software. The extractedzone(s) of sample material may include an analysis sample. Otherwise,the identified and extracted zone(s) can include zones which arediscarded and the remaining zone(s) which are retained on the glassslide 2, to be later analyzed.

[0043] In addition to the manual operation which is discussed in moredetail below, it is possible, according to another embodiment of thepresent invention, to utilize the image on the display monitor 4 toselect and identify a sample zone(s) whose relative position isdetermined by utilizing a computer which receives a digitized signal ofthe image from the video camera (or microscope), and which receives areference position of the stage of the microscope 3 upon which thesample is held.

[0044] In this automated embodiment of the invention, the computer whichperforms the positioning detection and recognizing can also be used tocontrol movement of the devices discussed below that are used to extracttissue zones, thus automating the sample removal. In addition, the imageof the sample can be electronically scanned to automatically identifyzones having a predetermined feature, such as a relevant degree ofstaining, using known techniques and devices. Thus, in a preferredembodiment, a computer could be used to select and identify zones ofinterest and the relative position of such zones, for manipulating adevice to remove such zones in a completely automated manner.

[0045]FIGS. 2a-2 c are a series of functional system diagrams which showhow a zone of tissue sample 1 is extracted from the slide-mounted tissuesample 1 according to one embodiment of the present invention. It is tobe understood that the steps depicted in FIGS. 2a-2 c could be eitherpreformed manually by an operator or by a computer utilizingconventional positioning and control methods, e.g. computer controlledrobotics.

[0046] The embodiment of the invention depicted in FIGS. 2a-2 c utilizea contact probe 5 which has an adhesive/extraction reagent 6 on the tipthereof. A suitable adhesive/extraction reagent can include a mixture ofpiccolyte and xylene. In FIG. 2a, the contact probe 5 is positionedeither manually or by computer control so as to be above and alignedwith the sample zone (“B”) to be extracted. As can be readily understoodfrom FIG. 2a, the surface area of the contact probe tip (andadhesive/extraction reagent) needs to be about equal to, and no greaterthan, the surface area of the zone to be extracted. Otherwise, excessiveremoval of adjacent tissue zones will occur. Manufacture of probe tipsof the required size is well within the capabilities of those skilled inthe art.

[0047] Once the tip of the contact probe 5 is aligned with the samplezone (“B”) to be extracted, the contact probe 5 is lowered so that theadhesive/extraction reagent 6 on the tip thereof contacts the samplezone (FIG. 2b). Of course, depending on the specifics of the apparatus,the probe 5 is raised or otherwise moved into contact with the samplezone of cells of interest.

[0048] The adhesive/extraction reagent 6 is selected to readily adhereto the sample zone. Once the adhesive/extraction reagent 6 on the tip ofthe contact probe 5 contacts the sample zone (FIG. 2b) and the samplezone becomes adhered thereto, the contact probe 5 can be retracted fromthe contact position (illustrated in FIG. 2b) and moved as shown in FIG.2c. Since the relative adhesive force of the adhesive/extraction reagentis greater than the adhesive force used to mount the sample on the glassslide, the contact probe 5 pulls the sample zone “B” from the glassslide when withdrawn or retracted.

[0049] According to one embodiment of the present invention, a glasspipette was used as the contact probe 5. In this embodiment, the tip ofthe glass pipette was coated with a solution of piccolyte (568 g/l) andxylene (437.5 g/l) by dipping the tip of the glass pipette in apiccolyte/xylene solution.

[0050] In addition to removing the sample zone from the glass slide 2,the contact probe 5 can be used to transfer the extracted sample zone toan analysis container 7 as indicated in FIG. 2c or to any otherlocation, such as a waste container, a culture media, etc. In apreferred embodiment, the contract probe 5 is used to transfer theextracted sample zone to the sample receiving stage of an automatedclinical analyzer which is designed to preform a desired analysis of thesample zone. It thus should be understood that the present invention canprovide a fully automated method for identifying sample zones on asample on a surface such as a slide, removing sample zones of interestfrom the surface-mounted sample, and transporting the extracted samplezones to an automated analyzer which can perform automated analysis ofthe extracted sample zones. Such analysis can include, for example,analysis of cellular DNA, RNA, proteins, polypeptides, lipids,carbohydrates, and combinations and aggregates thereof.

[0051] In FIG. 2c the extracted sample zone is depicted as beingdispensed in a container 7 which, for example, can be a test tube orsimilar container in which analysis on the extracted sample zone can beinitiated or performed. As depicted in FIG. 2c, a reagent solution 8which removes all or a desired component of the extracted sample zonefrom the contact probe tip can be placed in the container 7 before theextracted sample zone is deposited therein. For example, in the case ofDNA analysis, a solution of Tris (50 mM, pH8.5), EDTA (1 mM), Tween 20(0.5%), and proteinase K (0.2 mg/mL) can be used. This solution extractsthe sample zone from the tip of the contact probe 5 and dissolves thetissue material for analysis purposes.

[0052] In addition to the contact probe depicted in FIGS. 2a-2 c, ahollow suction probe could also be used to extract sample zones from theslide-mounted tissue sample 1. Such a suction probe could be providedwith sharp annular tip by which sample zones could be punched out andextracted by suction forces.

[0053]FIG. 3 is a schematic illustration of an alternative device forextracting sample zones from the slide-mounted tissue sample 1. Theextraction device 9 shown in FIG. 3 includes a cutting blade 10 and agrasping arm 11. The grasping arm 11 can be moved in an opposed mannerwith respect to the cutting blade 10. The grasping arm 11 is shown inits open position in FIG. 3. The grasping arm 11 is movable between theillustrated open position to a closed position in which the tip of thegrasping arm 11 contacts the cutting blade 10. The movement of thegrasping arm 11 can be controlled by a cable and pulley system in whichgrasping arm 11 is caused to pivot at its base by applying tension to acable which passes through a pulley located at the base of the graspingarm 11. The tension on the cable can be applied by actuating a lever ordepressing a button 12 on the device which applied tension to the cablein a known manner. Such actuating mechanical structures are known in theart of gripping devices.

[0054] In operating the device of FIG. 3, the cutting blade 10, which isat an obtuse angle with respect to the central axis of the device cancut out and scoop up a portion of a tissue sample by placing the cuttingblade 10 on one edge of a portion of the tissue sample to be extractedand then moving the grasping arm 11 into the closed position. As thegrasping arm 11 comes into contact with the tissue sample, it draws thecutting blade 10 into the sample and presses a portion of the sampletoward the cutting blade 10 thereby causing a portion of the samplecontacted between the cutting blade 10 and the grasping arm 11 to be cutout and scooped up from the sample.

[0055] In a further, alternative embodiment of the device of FIG. 3, themovement of the grasping arm 11 can be effected by a toothed gearinstead of a pulley and a cooperating toothed rod in place of a cable.Additional such mechanical structures are known in the art of grippingdevices.

[0056]FIGS. 4a and 4 b are schematic diagrams of a manual extractiontool manipulator which can be used together with the extraction deviceof FIG. 3 according to the present invention. In FIG. 4a, the extractiontool manipulator is depicted as having a base 13 equipped with aclamping means 14 for removably attaching the device to a brace orsupport portion of the stage of a microscope (see FIG. 4b). The clampingmechanism includes a clamping plate 15 that is secured to a threadedshaft 16 which passes through a threaded bore 17 in a lower portion ofthe base 13. A tightening knob 18 is provided on the end of the threadedshaft 16. Turning the tightening knob 18 causes the clamping plate 15 tomove with respect to an upper portion 19 of the base 13. Thus, theextraction tool manipulator can be clamped to a portion of the stage ofa microscope 20 as depicted in FIG. 4b by positioning a brace or supportportion 21 of the stage of the microscope 20 between the clamping plate15 and the upper portion 19 of the base 13 and turning knob 18 totighten the clamping plate 15 against the brace or support portion 21 ofthe stage of the microscope 20.

[0057] The extraction tool manipulator includes a tool holder 22 havinga through-bore 23 therein for receiving the shaft of an extraction tool24. Ideally, the tool holder 22 should allow for damped fore and aftmovement of the extraction tool. Therefore, according to a preferredembodiment, the through-bore 23 of the tool holder 22 contains a bushingwhich can be adjustably tightened against the tool shaft by tool lockingscrew 39.

[0058] The tool holder 22 is supported by support shaft 25 which isconnected at opposite ends by substantially 360° damped swivels 26 and27 to the tool holder 22 and the base 13. The length of the supportshaft 25 between the 360° damped swivels 26 and 27 is adjustable. Theadjustment of the independent 360° damped swivels 26 and 27 togetherwith the adjustable length of the support shaft 25 and the position ofthe tool shaft within through-bore 23, allows a high degree of movementof the extraction tool with respect to a slide-mounted sample positionedon the stage of the microscope. Therefore, an operator can manipulate anextraction tool held by the extraction tool manipulator and removeselected tissue zones from a slide-mounted tissue sample with a highdegree of precision.

[0059]FIG. 5 is a functional system diagram which shows how a zone ofsample tissue can be directed to an appropriate analysis protocol. Asdepicted in FIG. 5, a microextraction of a zone of tissue sample can betaken from a slide-mounted tissue sample 1 as discussed above andtransferred to a sample preparation stage 28 in which the cells ofinterest can be extracted and collected for analysis. Excised cells mayalso be solubilized at this stage. If these cells contain, or aresuspected to contain, one or more DNA or RNA of interest, the extractedsample may be subjected to polymerase chain reaction (PCR)amplification, followed by, for example, hybridization, strandconformational polymorphism, and southern and northern blotting,sequencing, etc. as desired. Of course, other techniques for analysis ofDNA and RNA are known to those skilled in the art and encompassed by thespirit and scope of the invention.

[0060] If the extracted cells contain, or are suspected to containproteins or polypeptides of interest, the extracted sample can besubjected to enzyme zymography, for example using one or more labeledsubstrates, an immunoassay utilizing, for example, labeled antibodies orfunctional fragments thereof, a biochemical assay, and the like.

[0061] Selective extraction or microdissection of frozen tissue sectionsaccording to the present invention allows for recovery and analysis ofboth active enzymes and mRNA. Additionally, the DNA recovered from thesesections is in the native condition and can be used for studies such asDNA fingerprinting. Microdissection of paraffin embedded tissuesaccording to the present invention allows for PCR amplification of DNA,for example, from pure cell populations representing less than one highpowered field, or a single layer of epithelial cells lining cysticspaces.

[0062] For general preparation of samples for frozen sectionmicrodissection according to the present invention, microdissectionslides can be prepared by placing 1% agarose on a standard histologyslide and cover slipping. After a short period of time, e.g., about 5minutes, the cover slip is removed leaving a thin gel on the slide. Asmall frozen tissue section, e.g. about 25 micron thick, is placed onthe agarose gel and briefly stained with eosin. The tissue may also betreated with agents to denature or otherwise inhibit RNase depending onthe subsequent extraction method. Under direct microscopicvisualization, the specific cell population or sub-population ofinterest is procured from the tissue section utilizing the techniquesdiscussed above.

[0063] For enzyme analysis the procured tissue specimen can be placed inan appropriate buffer depending on the enzyme of interest, as known tothe person skilled in the art. The enzyme levels can be measured byseveral methods including zymography and the use of specific substrates,including fluorometric, colorometric and radioactive substrates. Theprecise levels of enzyme expression in a specific, predefined cellpopulation can be thus determined and, where desired, compared to thatof another, independently isolated sample from the tissue sample.

[0064] For mRNA analysis the tissue specimen can be placed on agaroseand treated with agents to denature or otherwise inhibit RNase, ifdesired. The procured tissue specimen is immediately frozen in liquidnitrogen. The tissue can be used immediately or stored at −70° C. forseveral months. The mRNA can be extracted using, for example, columnchromatography on oligo-dT (Micro-FastTrack mRNA Isolation Kit,Invitrogen Co.). The recovered mRNA of the pure cell populations canalso be amplified and investigated using polymerase chain reaction (PCR)technology, such as, for example, by RT-PCR as known to those skilled inthe art.

[0065] For DNA analysis the tissue specimen can be placed in a singlestep extraction buffer solution of 50 mM Tris, pH 8.5, 1 mM EDTA, 0.5%Tween 20, and 0.2 mg/ml proteinase K, incubated for four hours at about37° C., followed by ten minutes incubation at about 95° C. The recoveredDNA can also be amplified and analyzed using PCR technology incombination with analysis techniques, such as blotting, sequencing,etc., known in the art. If native DNA is required for DNA fingerprintinganalysis, the proteinase K can be added after DNase in thefingerprinting protocol.

[0066] For paraffin section microdissection routine, formalin fixed,paraffin embedded tissue sections are microdissected afterde-paraffinization and brief staining with eosin. Tissue sections arevisualized by direct microscopy and cell populations or subpopulationsof interest are procured using a modified glass pipette with theadhesive coated tip discussed above. Tissue specimens as small as onecell can be procured with this method. The specificity of dissectionrepresents a significant improvement over currently known techniques.

[0067] For DNA analysis of paraffin embedded tissue, the glass pipettewith the dissected tissue specimen is placed in a single step extractionbuffer solution of 50 mM Tris, pH 8.5, 1 mM EDTA, 0.5% Tween 20, and 0.2mg/ml proteinase K, which removes the tissue from the pipette tip. Thesample is incubated, depending on sample size, from two to twenty-fourhours at about 37° C., followed by a ten minute incubation at about 95°C. The glass pipette tip can then be sterilized and reused, althoughthis is not generally recommended in the case of PCR-based analysis dueto the potential amplification of cross-contaminating materials.

[0068] According to the general procedure, an adhesive surface is placedin contact with the surface of the cells or tissue and the adhesiveforce binds the cellular material of interest to the adhesive surface.The adhesive surface, which can be the tip of a tool or needle, is usedto procure the material and transfer it to a liquid analysis reactionmixture. Examples of adhesive surfaces include adhesive coatings on thetip of the tool, or the use of electrostatic forces between the tip andthe surface of the cellular material.

[0069] As described in detail below, the isolation and transfer methodsof the present invention can involve a specialized continuousactivatable adhesive layer or surface which is applied to the cellularmaterial over an area larger than the area selected for microscopicprocurement. The adhesive function of the subsection of the surface incontact with the area selected for procurement is activated byelectromagnetic or radiation means.

[0070]FIGS. 8a-8 d are schematic illustrations of the sequential stepsof an adhesive transfer method according to one embodiment of thepresent invention.

[0071] As depicted in FIG. 8a, the adhesive transfer method utilizes atransfer surface 30 which includes a backing layer 31 and an activatableadhesive layer 32. In procedures which utilize laser activation of theadhesive layer, the backing layer 31 is preferably transparent, e.g.made of a transparent polymer, glass, or similar material. Theactivatable adhesive layer 32 can be an emulsion layer, a coated film,or a separate impregnated web fixed to the backing layer. Examples ofmaterials from which the adhesive layer 32 can be made include thermalsensitive adhesives and waxes (e.g., #HAL-2 180C from PrecisionCoatings), hot glues and sealants (available from Bay Fastening Systems,Brooklyn, N.Y.), ultraviolet sensitive or curing optical adhesives(e.g., N060-NOA81, ThorLabs Inc.), and thermal or optical emulsions(e.g., silkscreen coated emulsion B6 Hi Mesh, Riso Kagaku Corp.)

[0072] The backing layer 31 provides physical support for the adhesivesurface, and thus can be integrated physically into the activatableadhesive surface.

[0073] The activatable adhesive layer 32 is characterized by its abilityto be stimulated (activated) by electromagnetic radiation so as tobecome locally adherent to the tissue. For purposes of selectivelyactivating the activatable adhesive layer 32, one or more chemicalcomponents can be incorporated into the layer, which chemical componentscause selective absorbance of electromagnetic energy. Preferably, suchchemical components are IR-absorbable dyes suitable for use inconjunction with, for example, laser diodes.

[0074] As depicted in FIG. 8a, the transfer surface 30 is initiallypositioned over a cellular material sample 33 which can be a microtomesection or cell smear which is supported on a support member 34 such asa microscopic slide. In the case of a tissue microtome, routineprocedures can be used to provide paraffin-embedded, formalin-fixedtissue samples.

[0075] As shown in FIG. 8b, the transfer surface 30 is brought intocontact with the cellular material sample 33. It is noted that theactivatable adhesive layer 32 preferably has a larger area than thesubregion of cellular material sample which is subsequently selected forprocurement.

[0076] The transfer surface 30 can be fixed to the cellular materialsample support 33 by clips, guides, tape, standard adhesives, or similarconvenient means. The transfer surface 30 can also contain a labelregion 35 (see phantom lines in FIG. 8b) to write information such asthe patient's identification code or a test designation.

[0077] After the transfer surface 30 is brought into contact with thecellular material sample 33, the cellular material sample is viewed bystandard low or high power microscopy to locate the region of interest.This region can range in size from an area smaller than a single cell(less than 10 microns), to a few cells, to a whole field of cells ortissue. When the area of interest is identified, the precise region ofthe activatable adhesive layer 32 which is immediately above the area ofinterest is activated by a beam of electromagnetic energy 36, e.g., alaser beam, as depicted in FIG. 8c.

[0078] Application of the electromagnetic energy 36 causes the region ofthe activatable adhesive layer 32 which is immediately above the area ofinterest to adhere to the area of interest. Although FIGS. 8c and 8 ddepict a single region of interest, it is to be understood thatmultiple, discontinuous regions of interest could be selected andprocured by appropriate aiming and application of the electromagneticenergy.

[0079] As depicted in FIG. 8d, after one or more regions of interest “A”are identified and the corresponding region(s) of the activatableadhesive layer 32 is activated by a beam of electromagnetic energy 36,the transfer surface 30 is detached from the cellular material samplesupport 34. As shown, the removed transfer surface 30 carries with itonly the precise cellular material from the region of interest “A”,which is pulled away from the remaining cellular material sample.

[0080] As mentioned above, a single transfer surface can be used toremove a plurality of areas of interest from a single cellular materialsample. The transfer surface 30 carrying the procured cellular materialcan be treated with suitable reagents to analyze the constituents of thetransferred material. This can be accomplished by submerging thetransfer surface 30, to which the procured cellular material is adhered,in a suitable reagent solution. Alternatively, one or more of theprocured cellular material regions can be removed from the transfersurface 30, or portions of the transfer surface 30 to which the procuredcellular material are adhered can be punched out of the transfer surface30 and analyzed separately.

[0081] In another embodiment of the invention, one or more cells ofinterest are isolated via laser capture microdissection as schematicallydepicted in FIG. 9. In this method of the invention, a tissue samplespecimen 50 is mounted on a support, such as glass slide 52. As before,a transparent or translucent film or tape 54 (the transfer film) isplaced on top of the tissue sample specimen 50. The tissue sample 50 isthen examined microscopically for predetermined target cells, such asabnormal cells (or control cells for comparison). As before, the cellsmay be stained with dye(s), immunologically, etc. to identify and/ordifferentiate the predetermined cells 56 of interest in the sample. Thepredetermined cells of interest 56 are next made to coincide with atarget point, wherein electromagnetic radiation may be focused. Thiscoincidence may be accomplished, for example, by x-y-z translation ofeither the specimen 50 or the target point. For example, the targetpoint may coincide with the center of the imaging field and themicroscope stag, translated such that the predetermined cells ofinterest are brought to this target point. One or more focused pulses ofenergy (e.g., electromagnetic energy in the form of light from a laser)is directed at the film overlying the target. Sufficient energy isdirected to the target point so as to preferentially heat or otherwisealter the adhesive characteristics of the film or tape covering thepredetermined cells of interest at the target point. In this way, thefilm or tape 54 is made selectively adhesive at the specific target 56in the sample by optical activation in precisely predefined locations.

[0082] It is preferred that lasers 58 are used in the present inventionwhich, in conjunction with lens 60 and mirror 62 provide electromagneticenergy to the target spot. This is because lasers 58 are high brightnesslight sources of intense, collimated light that can be readily andefficiently focused to small regions on a given surface. By using alaser focus onto the optical center of the field of view of an opticalmicroscope, activation energy can be supplied to a target region of thefilm or tape lying on top of the tissue sample. Moreover, the timing andduration of lasers are readily controlled, such that a controlled amountof energy can be directed to the target spot. Additionally, a laser beamcan be focused to spots as small as the diffraction limit of thewavelength used and thus permit selective adhesion to targets as smallas one micron. Thus, the spots can be small enough to select ahomogenous cluster of cells, an individual cell, or even a portion of acell.

[0083] When sufficient energy from the focused pulse of radiation isabsorbed to provide activation of the film surface 54 which is incontact with the predetermined cells of interest in the tissue sample,an adhesive bond is formed between the film or tape 54 and thespecifically targeted cells 56. As long as the focal bond strengthformed between the film 54 and the targeted tissue 56 is greater thanthe bond strength for the targeted tissue for the underlying substrate(e.g., microscope slide 52 and the bond strength within the targetedtissue), the targeted tissue 56 can be procured upon the removal of thefilm.

[0084] The size of the tissue transferred, depending upon the needs ofthe operator, can be varied by changing the diameter of the laser beamand pulse durations. Highly reproducible transfers in the 60 to 700 μmdiameter range are easily attainable for procurement of small (100 μm to1 mm) lesions without the encroachment of adjacent, nonneoplastic cells.In most basic and clinical research studies, procurement of severalhundred to several thousand cells is necessary to provide sufficientgenetic material for reliable amplification and statistically meaningfulanalysis. However, since laser beams can be focused to less than a onecell diameter, transfers of targeted single cells or even parts thereofis thought possible under the practice of the invention.

[0085] Thermoplastic polymer films are widely used as heat and pressureactivated adhesives for bonding surfaces. Most of these polymer filmsare transparent or translucent to visible light used in conventionallight microscopy. These films are, however, strongly absorptive inspecific regions of the electromagnetic spectrum (e.g., in regions ofthe infrared associated with strong molecular vibration modes such as3000, 1800, 1400-960 cm⁻¹)

[0086] The transfer film 54 may be made of a wide variety ofelectromagnetically or thermally activatable materials, such as ethylenevinyl acetate (EVA), polyurethanes, polyvinyl acetates, and the like.Specific other selectively activatable materials found useful in thepractice of the invention are: thermal sensitive adhesives and waxes,such as Precision Coatings product #HAL-2 180C; thermally-activated hotglues and sealants, such as those from Ban Fastening Systems (Brooklyn,N.Y.); ultraviolet sensitive or curing optical adhesives, such asThorLabs, Inc. product N060-NOA81; thermal or optical emulsions, such assilkscreen coated emulsion B6, high mesh powdered, reconstituted leltfixit emulsion (Riso Kagaku Corp.) and various other compounds includingacetal, acrylic, alloys and blends, allyl, bismaleimides, cellulosics,epoxy, fluoroplastics, ketone-based resins, liquid crystal polymers,melamine-formaldehyde, nitrile, nylon, phenolic, polyamide,polyacrylate, polybenzimidazole, polybutylene, polycarbonate,thermoplastic polyester, liquid crystal polymers, polybutyleneterephthalate (PBT), polycyclohexvlenedimethylene terephthalate (PCT),engineering grade polyethylene terephthalate (PET), standard gradepolyethylene terephthalate (PET), thermoset polyetherimide polyethylenepolyester, branched polyethylene, ethylene acid copolymer,ethylene-ethyl acrylate (EEA), ethylene-methyl acrylate (EMAC),ethylene-vinyl alcohol copolymers (EVOH), high-density polyethylene,HMW-high-density polyethylene, Ionomer, linear low-density polyethylene,linear polyethylene, low-density polyethylene, UHMW polyethylene, verylow-density polyethylene, thermoplastic polyimide, thermoset polyimide,polymethylpentene, modified Polyphenylene oxide, polyphenylene sulfide,blow molding PPS, polyphthalamide, polypropylene, polypropylenehomopolymer, polypropylene impact copolymers, polypropylene randomcopolymers, silicones, styrenic resins, ADS, ACS,acrylic-styrene-acrylonitrile, expandable polystyrene, general purposepolystyrene, impact polystyrene, olefin-modified SAN, polystyrene,styrene-acrylonitrile (SAN) and styrene-butadiene copolymers.

[0087] The adhesive film described above may be self-supporting orlaminated with a support film. Additionally, the support film may bemade of a material that does not absorb the electromagnetic energy sostrongly as to interfere substantially with the activation of thethermoplastic polymer. The support film preferably absorbs weakly, if atall, at the activation wavelength and at the visualization wavelength.The activatable film, on the other hand, preferably absorbs weakly, ifat all, at the visualization wavelength but strongly at the activationwavelength. The support film should also be unaffected by the resultingthermal transients occurring during the activation.

[0088] It is also possible to add infrared absorbing dyes to thethermoplastic films 54 to provide strong absorption at other specificinfrared wavelengths without altering the films transparency to visiblelight. Such dyes are preferably IR absorbing dyes which are readilysoluble in the plastic films and have a very strong, narrow IR or nearIR absorption bands that can be matched to a variety of IR or near IRlasers (including particularly laser diodes). If the focused pulse ofelectromagnetic radiation (e.g., laser) is delivered at wavelengths thatare strongly absorbed by the film, then the film may be efficientlyfocally heated.

[0089] Many dye types could be considered for IR absorption, since mostclasses of visible absorbing dyes can be extended in wavelength bymolecular modification. Phthalocyanines and cyanines have been among themost popular dyes because of stability, ease of preparation, solubility,optical and other properties. Moreover, the number of possiblemodifications of these dyes is very large because various central metalatoms which can be added and a variety of ring attachments which can bemade to them. A book which gives a general overview of IR absorbing dyesis:

[0090] INFRARED ABSORBING DYES

[0091] Masaru Matsuoka, ed. (U. of Osaka, Sakai, Osaka)

[0092] Plenum Press NY 1990 0-30843478-4

[0093] TA1690.I53 1990 available in the NBS library series: Topics inApplied Chemistry, A. R. Katritzky and G. J. Sabong, eds.

[0094] As an example of phthalocyanine dyes, the following 60 entriesare in the Aldrich Chemical Catalog: TABLE 1 PHTALOCYANINE DYES (AldrichChemical Company) #412066 Name: TETRAKIS(4-CUMYLPHENOXY)PHTHALOCYANINE,97% #404543 Name: TIN(II) PHTHALOCYANINE #406481 Name: SILICONPHTHALOCYANINE DIHYDROXIDE Cata #414387 Name: VANADYL2,9,16,23-TETRAPHENOXY-29H,31H- PHTHALOCYANINE #393932 Name:MANGANESE(III) PHTHALOCYANINE CHLORIDE #410160 Name: IRON(II)PHTHALOCYANINE BIS(PYRIDINE) COMPLEX #404551 Name: TITANYLPHTHALOCYANINE #418145 Name:1,8,15,22-TETRAPHENOXY-29H,31H-PHTHALOCYANINE #418153 Name:2,9,16,23-TETRAPHENOXY-29H,31H-PHTHALOCYANINE #379573 Name: IRON(III)PHTHALOCYANINE CHLORIDE Catal #406473 Name: TIN(IV) PHTHALOCYANINEDICHLORIDE Catal #415448 Name: NICKEL(II) TETRAKIS(4-CUMYLPHENOXY)PHTHALOCYANINE #418161 Name:1,8,15,22-TETRAKIS(PHENYLTHIO)-29H,31H- PHTHALOCYANINE #418188 Name:2,9,16,23-TETRAKIS(PHENYLTHIO)-29H,31H- PHTHALOCYANINE #408808 Name:GALLIUM(III) PHTHALOCYANINE CHLORIDE Ca #418986 Name: ALUMINUM2,9,16,23-TETRAPHENOXY-29H,31H- PHTHALOCYANIN #310204 Name: COPPER(II)4,4′,4″,4′″-TETRAAZA-29H,31H- PHTHALOCYAN #402737 Name: MAGNESIUMPHTHALOCYANINE #402745 Name: DISODIUM PHTHALOCYANINE #418250 Name:ALUMINUM 2,9,1 #341169 Name: ZINC PHTHALOCYANINE #379557 Name:MANGANESE(II) PHTHALOCYANINE Catalog Nu #414379 Name: NICKEL(II)2,9,16,23-TETRAPHENOXY-29H,31H- PHTHALOCYAN #433462 Name: METHYLSILICON(#418234 Name: ZINC 2,9,16,23-TETRAKIS(PHENYLTHIO)-29H,31H- PHTHALOCY#418242 Name: ALUMINUM 1,8,1 #379549 Name: IRON(II) PHTHALOCYANINE#408875 Name: LEAD(II) TETRAKIS(4-CUMYLPHENOXY)PHTHALOCYANINE #393894Name: VANADYL 3,10,17,24-TETRA-TERT-BUTYL-1,8,15,22- TETRAKI #432946Name: COPPER(II) TET #441082 Name: GALLIUM(III) P #423157 Name:2,9,16,23-TETR #393886 Name: COPPER(II) 3,10,17,24-TETRA-TERT-BUTYL-1,8,15,22-TETR #418269 Name: ALUMINUM 1,8,1 #423165 Name: COPPER(II) 2,9#430994 Name: ZINC 2,9,16,23 #307696 Name: COBALT(II) PHTHALOCYANINECatalog Number #432180 Name: SILICON 2,9,16 #446637 Name: ALUMINUM PHTHA#253103 Name: 29H,31H-PHTHALOCYANINE, 98% Catalog Num #379565 Name:LEAD(II) PHTHALOCYANINE #418277 Name: ALUMINUM 2,9,1 #362530 Name:ALUMINUM PHTHALOCYANINE CHLORIDE Catalo #444529 Name: ZINC 1,2,3,4,8#452521 Name: IRON(III) PHTH #446645 Name: COBALT(II) 1,2 #446653 Name:COPPER(II) 1,2 #448044 Name: IRON(II) 1,2,3 #386626 Name: ALUMINUM1,4,8,11,15,18,22,25-OCTABUTOXY- 29H,31H-PHTH #360635 Name: NICKEL(II)PHTHALOCYANINE Catalog Number #448311 Name: COPPER(II) 1,2 #428159 Name:SILICON(IV) PH #386618 Name: COPPER(II) 1,4,8,11,15,18,22,25-OCTABUTOXY-29H,31H-PH #287768 Name: SILICON PHTHALOCYANINE DICHLORIDE Catal #408883Name: NICKEL(II) 1,4,8,11,15,18,22,25-OCTABUTOXY- 29H,31H-PH #383813Name: ZINC 1,4,8,11,15,18,22,25-OCTABUTOXY-29H,31H- PHTHALOC #362549Name: DILITHIUM PHTHALOCYANINE #383805 Name:1,4,8,11,15,18,22,25-OCTABUTOXY-29H,31H- PHTHALOCYANIN #252980 Name:COPPER(II) PHTHALOCYANINE Catalog Number #245356 Name: COPPER(II) PHT

[0095] An example of a traditional near-IR absorbing dye, also used fordiagnostic purposes, is Aldrich #22886-9 dye, indocyanine green.Another, used as a biological stain, is Aldrich #11991-1, naphthol greenB. Of all these dyes, a particularly good choice for this applicationare the naphthalocyanine dyes which have low water solubility but highsolubility in non-polar polymers. For example, vanadyl5,14,23,32-tetraphenyl 2,3-naphthalocyanine [Aldrich 39,317-7 (CA131220-68-3)] with a molecular formula weight of 1084 daltons exhibits astrong absorption peak (with a molar extinction coefficient of^(˜)200,000 at 846nm) and high solubility in ethylene vinyl acetate(EVA) low melting polymers (such as Dupont ELVAX™ 410). This dyeabsorption peak matches well the emission wavelength of selected GaAlAslaser diodes. Similarly, vanadyl2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine [CA 105011-00-5] FW1004absorbs near IR with a narrow peak at 808 nm which closely matches theemission wavelength (selected by choosing a different value of [Al]) ofGaAlAs laser diodes widely used to pump solid state Nd:YAG lasers. Allthese naphthalocyanine dyes (Table 2) are highly soluble in EVA polymerthermoplastics and other similar thermoplastic materials. They are quitestable compounds particularly with heating to ^(˜)300° C. and do notexhibit adverse photochemistry which might affect biologicalmacromolecules in the tissue.

[0096] A table of naphthalocyanine dyes as presented in the AldrichCatalog is presented below: TABLE 2 NAPHTHALOCYANINE DYES (AldrichChemical Company) 1) vanadyl 5,14,23,32-tetraphenyl 2,3-naphthalocyanineAldrich 39,317-7CA 131220-68-3FW1084 846 nm p. 104 2) tin(IV)2,3-naphthalocyanine dichloride Aldrich 40,651-1CA 26857-61-4 FW902 828nm p. 102 3) silicon (IV) 2,3-naphthalocyanine dihydroxide Aldrich40,653-8CA 92396-90-2 FW775 785 nm p. 94 4) silicon(IV)2,3-naphthalocyanine dioctyloxide Aldrich 40,767-4CA 92941-50-9 FW941798 nm p. 94 5) 5,9,14,18,23,27,32,36-octabutoxy 2,3-naphthalocyanineAldrich 41,207-4CA 105528-25-4FW1292 867 nm p. 181 6) copper(II)5,91,14,18,23,27,32,36-octabutoxy 2,3- naphthalocyanine Aldrich41,528-6CA 155773-67-4FW 853 nm p. 33 7) nickel(II)5,9,14,18,23,27,32,36-octabutoxy-2,3- naphthalocyanine Aldrich41,885-4CA 155773-70-9FW1348 848 nm p. 78 8) vanadyl2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine Aldrich 43,296-2CA105011-00-5FW1004 808 nm p. 1524 ′96

[0097] A variety of wavelengths of electromagnetic energy can be used inthe practice of the invention provided that suitable materials are used.In particular, it is important that the transfer film 54 absorbsufficient energy (or contains one or more dyes that absorb sufficientenergy) at the chosen wavelength to melt or nearly melt thethermoplastic polymer in the targeted region. For thermoplasticmaterials such as ethylene vinyl acetate (EVA), a wavelength of about 3to about 10 micrometers is preferred as these materials intrinsicallyabsorb in this range. In one embodiment, the power of the laser is usedgenerally in the range of from about 1 mW to about 200 mW depending onthe size of the target (i.e, increasing power with increasing targetsize). It is also preferred that the wavelengths for laser activationand film absorption be chosen outside the normal range used formicroscopic imaging. Reproducible microtransfer of tissue can beobtained using a variety of infrared wavelengths from the laser.

[0098] Suitable lasers for use in the present invention include carbondioxide lasers (9.6-11 μm wavelengths), laser diodes, tunable singlefrequency Ti:sapphire lasers and diode-pumped NdYAG lasers. Thewavelength outputs from these lasers can preferably range fromultraviolet to infrared. A particularly desirable laser for use with thepresent invention is the laser diode with wavelengths between 690 and1300 nm. In this wavelength range, conventional glass microscope opticsis highly transmissive and can be used to focus the laser. Othermaterials such as clear plastic support layers (e.g., polyester) for thethermoplastic film or pressure plates made of glass or plastic are alsohighly transmissive (with very low absorption) and therefore can be usedeasily. This is a marked improvement over laser capture microdissectiondesigns using longer laser wavelengths (e.g., 9.6-11 μm) for the carbondioxide laser or ^(˜)3um with the Er:YAG with laser diode pumping orintrinsic).

[0099] As shown in FIGS. 10A and 10B, the use of a compact laser diode58 allows the laser 58 to be easily incorporated into a conventionalmicroscope housing. For example, in FIG. 10A, incandescent light 66 fromthe microscope can be used, through a lens 68 and dichroic mirror 62, toidentify and focus on the tissue which is desired to be extracted. Lightfrom the laser diode 58 can then be focused through lens 60 and dichroicmirror 62 along the same effective optical path to allow readydissection of the tissue which has been identified through themicroscope. This “epi-irradiation” approach provides the advantages ofusing the same optics (microscope objective) to project high qualityimaging of the specimen and also to focus the laser beam to small spots.The economy of using high quality microscope objectives for this dualfunction simplifies access to the sample 54. In general, higher imagingmagnification will be coupled to smaller, higher precision laser capturemicrodissection targeting in this configuration.

[0100] By contrast, an advantage of the previously disclosed lasercapture microdissection configuration shown in FIG. 9 is that imagingmagnification and target spot size are decoupled and therefore moreeasily independently varied. In this later case, a targeting decisioncould easily be based on a high resolution image and then a simpleadjustment of the laser spot made to match the target size. With the“epi-irradiation” design of FIG. 10A, such adjustments would be mosteasily accomplished by either multiple pulses or by switching (swivel)the microscope objectives after imaging to give appropriate spot size.

[0101] The configuration of FIG. 10B addresses the problem ofeffectively projecting the heat provided by laser 70. More specifically,in the configurations of FIGS. 9 and 10A, the laser first irradiates thetop surface of a thermoplastic polymer film 54. In this case, the top ofthe film 54 absorbs a significant fraction of the laser pulse and isheated more rapidly than the lower surface. Consequently this topsurface of the film 54 melts more rapidly. In this configuration, theintrinsic spot size is limited by the film thickness and thermaldiffusional broadening of the heated spot of the film at the tissuespecimen surface. By projecting the laser light through lens 72 andmirror 76 onto the bottom surface of the film 65, the laser capturemicrodissection can be performed on much smaller target spots (as smallas 4 um) and with much shorter pulses (as short as 5 msec) to result inmore precise targeting of small components (e.g., single cells or cellnuclei) and exposing them to briefer thermal transients. The later isimportant in quantitative recovery of molecular function, particularlyenzymatic activity within the targeted cells, which is important insubsequent molecular analysis that is performed with tissue procured bythe laser capture microdissection.

[0102] Indicators may also be included, either in the selectivelyadhesive transfer film or in a separate layer or layers, to define thelocation of the optical activation. Such indicators includethermochromic dyes, dye precursors which combine upon melting to form acolor for visible or instrumental identification, and dyes which areconverted to color by other effects of optical absorption. Suitableindicators also include other physical effects, such as the appearanceor disappearance of translucency or opacity upon optical exposure orupon heating.

[0103] While wishing not to be bound by theory, it is thought that whensuch thermoplastic films are heated to near or at the melting point theyflow and conform to an adjacent surface (in this case, the targetedtissue sample), forming a strong surface bond. This bond is thought tooccur without actual chemical cross-linking to the tissue sample. Suchstrong bonds are formed most reliably when pressure is applied to forcethe flow of the “melt” into tight conformity with the sample surfaces.However, by using smooth films applied in close apposition to the tissueand delivering appropriate pulse parameters to selected compositionthermoplastic films, one can reliability heat the film to peaktemperatures associated with high film fluidity for a sufficient periodof time to form adequately strong bonds between the film and tissue forhighly reproducible focal microtransfer to occur. Moreover, by using apulsed infrared laser source to activate the focal bond of the targetedtissue to the film, the targeted tissue is quantitatively procured(virtually complete microtransfer to the film) without chemicalmodification, while preserving focal tissue morphology and allowingunaltered microscopic observation prior to, during, and following themicrotransfer.

[0104] An optional additional step may also be used in laser capturemicrodissection to improve the bonding between the cells of interest andthe activated polymer film and decrease the bonding of the tissue ofinterest to the substrate. For example, the slide can be chosen to be amaterial that has an inherently lower affinity for the tissue samplethan the polymer film has for the tissue sample. Alternatively, theslide can be pretreated with a monolayer of silicone oil or 3% aqueousglycerol solution to reduce the adhesion of the tissue subsequentlyplaced on the slide and increase the ease with which the activated filmremoves the tissue. In another variant, this tissue or film can becoated with a “release agent” such as silicone oil or a directly bondedsilicone polymer. In this way, the film will exhibit no affinity for thetissue in regions which are in contact with the tissue but not thermally(laser) activated. Thermal activation will cause focal melting of thebulk film polymer and thus disrupt and dilute the surface monolayer ofrelease agent in the region to be bonded. Therefore, the targeted tissuebond strength is unaffected by the release agent. In a fourth example,an adhesive layer such as poly-L-lysine can be coated onto the slidebefore application of the tissue section. This adhesion strengthens theattachment of the tissue to the slide, thereby reducing non-specifictransfer when the film is pressed onto the tissue before laseractivation. Although many such treatments may make specific transfermore difficult (i.e., requiring greater bond strength of the film withthe underlying tissue), an adhesive coating such as poly-L-lysine thathas a high affinity for and forms a strong bond with the meltedthermoplastic film may inhibit non-specific transfer while facilitatingcomplete specific transfer of the targeted regions. Finally, in a fifthexample, the tissue can be enclosed in a polymeric material which willform a strong bond with the meltable film and be sufficientlywater-soluble to allow the tissue sample to be retrieved in the analysisstep. The enclosure of the tissue in such a material can be done by acoating technique such as application of the polymer in solution orplacing the material in film form on the tissue and melting it. It couldalso be in the form of a coating on the hot-melt film to enhance thebond of the meltable film to the tissue and decrease the bond to theslide.

[0105] The laser microdissection system can be advantageously used witha variety of sample preparations, such as stained thin sections oftissue or stained cytology specimens of intact cells. In this case, thetransferred regions can be clearly identified in a microscope by thefocally transferred stained material on an otherwise transparent film.Alternatively, the film and tissue slide can be indexed to an x-ycoordinate system to give a specific slide location to each transferredpoint which may then be automatically recorded.

[0106] The microtransferred tissue may then be collected from the film,for example, by punching the precisely recorded spots directly into thedesired reaction or extraction vessels (e.g., by automatic x-ytranslation) or by placing the whole film into a reaction vessel.

[0107] The molecular analysis of the extracted cellular material, forexample by RT-PCR, in one embodiment of the invention requireslocalizing the small objects (e.g., 50 μm spots of tissue) adhering tothe substrate and collecting them into an analysis chamber, for example,punched out into a vial. Using an x-y encoding of the position of eachtarget site and automated translating allows the punching of area(s) toalso be automated using the same coordinates, so long as the supportfilm is not deformed or stretched in the application, activation andremoval steps of the process. Thus, the sample collecting process isalso amenable to automation. Alternatively, one can ensure that thetarget sites are at known positions on the transfer substrate. Forexample, multiple small pieces of adhesive transfer film can beselectively applied to those locations on the tissue which correspond tosites to be extracted, rather than applying a single large piece ofsubstrate film to the entire specimen. Exemplary of such a scheme issmall disks of adhesive film are applied at a fixed repeat distance on acontinuous polyester film, preferably a strong and not easily stretchedsheet, to provide a linear array of separately activatable target sites.After each target zone is identified, the next unused small adhesivespot in the linear array is locally applied at a fixed separation tothis region by a small pressure plate or an air jet. Thesubstrate/tissue slide as a unit is then micropositioned undermicroscope viewing to target specific cells (determined by the laserspot diameter) within the target zone (i.e., the diameter of theadhesive spots which is greater than the laser spot diameter). Where thesubstrate film and the mechanism applying these small pieces of film islocated with respect to a stationary reference (e.g., the microscopeobjective) then it can be arranged that the adherent tissue spots willalways be at known locations on the substrate film (e.g., in the centerof a narrow strip of adhesive film at equally spaced distances).Targeting of like cells within the target zone is accomplished bymicrotranslating the sample between-sequential laser pulses. Thus, theoperator positions the slide in the microscope so that the tissue ofpredetermined interest is at the center of the field, a pressure plateor other means can then adhere the adhesive film spot to the tissue suchthat the predetermined cells/objects of interest are within the knownlocation on the support film. The adhesive is activated, for example, bythe IR laser pulses, and the pressure plate is then released. The filmis then separated from the specimen slide and advanced so that a freshportion of film can be used at the next specimen location. The processallows all transfers to occur on an ordered series (numbered array) ofspots with a fixed spatial separation, as the film is not distorted inthe process. The size of the adhesive film spot determines the size ofthe target zone within which selection of the objects/cells are made forthat one transfer (array number) in this embodiment of the invention.The target zone size is determined by the selection of the particularfilm (i.e., the geometry of ordered arrays), but can be increased ordecreased, for example with parallel rows of spots of different sizes onthe same support film.

[0108] In another embodiment of the invention that is furthersimplified, the collection process is performed by cutting off (ratherthan punching out) the desired portions of adhesive films. Thissimplified embodiment eliminates the requirement for close tolerancesbetween a punch and die. Alternatively, other means can be used toseparate the tissue/adhesive film from the rest of the tape. Such meansto include direct peeling of the tape, focally dissolving either theadhesive tape or its bond to a substrate film, excising the spots with ahot wire knife (which is self-sterilizing to eliminate contaminationbetween specimens).

[0109] An additional feature of the invention is directed to theidentification of the samples. The small portions of film can haveminute identifying marks (e.g., bar codes) attached to them which wouldbe seen under the microscope and can be recorded along with a videoimage of the specimen. A practical way in which this is accomplished isto place the identifying marks on a mechanically strong substrateadjacent to each discrete spot of adhesive on the tape configurationmentioned above. An additional use of these identification marks is tocontrol the advance of the tape for each new specimen. A sensor in themicroscope or analysis of the various specimens determines when the markis in, for example, the center of the field as the tape is beingadvanced. Alternatively, a mechanical drive (e. g., with sprockets) canbe used to advance the tape a fixed known amount.

[0110] As a preferred embodiment of the invention, equally spacedadhesive film spots along with minute adjacent bar code identifiers arecentrally placed on a thin (e.g. 1 mm wide), mechanically strong backingtape, for example, about 0.002 inch thick mylar, which is preferablysupplied in a sterile cassette having a leader. The cassette and astepper motor takeup drive are attached to the housing of an invertedmicroscope (or its stationary stage if the slide is to be moved byhand), so that the center of the tape is aligned with the center of themicroscope objective (and field) and the tape is above the level of thespecimen. The leader is attached to a spool on the drive shaft of astepper motor and wound up enough so that the tape is firmly attached tothe takeup motor and the first adhesive spot is nominally in position. Asolenoid actuated pressure plate pushes down on the tape so that it isclose enough to the specimen that a sensor in the microscope (videosignal) can see the identifier marks before the tape is firmly attachedto the specimen. The tape is then advanced to its final position and thepressure place is pushed firmly against the film. An IR laser isactivated to bond the selected tissue to the adhesive film and shortlyafterwards the pressure plate is released. The pressure plate solenoid,which also holds a fixture with two prongs which lie between the filmand the stage is then temporarily actuated in the upwards direction sothat the two prongs pull the tape off of the tissue. A next specimen isoptionally selected by the operator and the pressure plate is activatedin its partial down position so that the sensor can detect theidentification mark and the process is repeated as desired. After thefinal specimen has been transferred, the motor advances the tape furtherand then the takeup spool and the cassette are removed and attached tothe motor shafts of a collection mechanism mounted on the stage. Thismechanism uses a hot wired knife to cut the adhesive spot/adherenttissue away from the rest of the tape and employs an air jet to separatethe two (if necessary) and deposit the sample into either a vial or a 96well microtitre plate. The known location of the adhesive spots alongthe length on the tape can be uses to properly position the first spotand advance the tape to subsequent positions. A computer-generated barcode correlated with the film label is attached to the vial ormicrotitre place for traceability. The bar code is stored in a computerdata entry of the microtransfer sample (e.g., images, patient number,specimen number, etc.) as well as being recorded directly in the imageof the target immediately after laser activation.

[0111] Laser capture microdissection (LCM) has many advantages over theprior art techniques. As an example of the benefits of the presentinvention, an individual glomerulus can be procured from a kidney tissuesection sample in under ten seconds, and hundreds of glomeruli can beisolated by a single operator in one hour with minimal effort. Oneskilled in the art appreciates that such speed and efficiency cannot byapproached by conventional microdissection methods.

[0112] It should also be appreciated that laser capture microdissectionis not limited to use on biological samples. Indeed, the techniquesdescribed herein may be used for the sorting/removal of any object thatneed be discriminated from other objects in a microscopic field. Forexample, micromachined objects can be readily, rapidly and efficientlysorted under the practice of the invention. It should further beappreciated that the practice of the invention is not strictly limitedto the use of electromagnetic energy as any energy source that providesfor a specific, localized melting of the thermoplastic transfer filmwill operate in the invention. A heat source, for example, from anelectrical circuit may be desirable when the region to be transferred issufficiently large as in, for example, a relatively homogeneous tissuesample on the order of 1 millimeter in size. Also useful as sources ofselective energy are electrically heated radiant heaters, irons orpencil heating probes, flashbulb generated energy (when used, forexample, in conjunction with one or more precision masks, focused xenonlamps, as obtainable from ILC Technology, Inc. of Sunnyvale, Calif., andthe like, as will be apparent to those skilled in the art based upon thedisclosure herein.

[0113] Features and characteristics of the present invention will beillustrated by the following examples to which the present invention isnot to be considered limited. In the examples and throughout,percentages are by weight unless otherwise indicated.

[0114] The following examples were performed in an attempt to establishif the present invention could be used to more specifically studyprotease distribution during human tumor invasion. Levels of MMP-2 andcathepsin B in fields of invasion breast and colon carcinoma weremeasured to assess if the enzymes in these regions were quantitativelyincreased as compared to matched numbers of normal cells from the samepatient.

[0115] In the following examples, normal and tumor samples of colon andbreast tissue from surgical resections were maintained in a frozencondition (−70° C.) until analysis. Tissue section of invasive breastand colon carcinoma were selected based upon histologic evaluation. Thetumor sections of tissue which contained invasive tumor and stroma werepreferentially selected instead of normal epithelium or significantnumbers of inflammatory cells. The control sections of normal tissuecontained epithelium and a thin section of underlying stroma. Theproportion of epithelial and stromal tissue was similar for both normaland tumor sections.

[0116] In the examples, microdissection slides were prepared by coveringstandard histology slides with 200 microliters of warm agarose (1%) andoverlaying a cover slip. After five minutes the coverslip was removedleaving a thin bed of agarose on the slide. Twenty micron thick frozensections were prepared in a cryostat and placed on the agarose gel. Thetissue was briefly dipped in eosin. Optimum microdissection was achievedby starting at the edge of each section and systematically dissectingand separating histologic fields of interest with the microdissectingdevice of FIG. 3. Areas of interest were retained on the slide forsubsequent analysis. The DNA content of the specimens was determined byspectrophotometric measurement at 260 nm. The DNA content of each samplewas proportional to the number of cells counted in each histologicsection.

[0117] cDNA (and DNA) libraries of microdissected tissue sections arealso provided for by the present invention as well as methods of makingsuch libraries. Such libraries are useful, inter alia, in facilitatingthe identification of transcripts specifically expressed in cells ofdistinct histological origin and tumorigenic stage.

EXAMPLE 1

[0118] In this example, samples of normal and tumor tissue matched forcell number were analyzed from each subject. Levels of MMP-2 weredetermined by zymography and quantified using an Arcus scanner. Resultswere statistically analyzed using the students t-test. Cathepsin Blevels were determined as V_(max), against the substrateZ-Arg-Arg-NHMec.

[0119] The results of this example are set forth in Table 1 below whichlists the cathepsin B activity in matched pairs of invasive coloncarcinoma/normal epithelium, and invasive breast carcinoma/normalepithelium. Activity measurement are expressed as V_(max), nmol/min×mgDNA. Cathepsin B activity was increased an average of 2.3 fold in thecolon tumors (p<0.005), and 6.9 fold in the breast tumors (p=0.077).TABLE 1 SAMPLE NORMAL TUMOR TUMOR/NORMAL CATHEPSIN B ACTIVITY ININVASIVE HUMAN COLON CARCINOMA 1 1.38 4.75 3.4 2 1.89 2.25 1.2 3 1.986.32 3.2 4 0.49 1.88 3.8 5 0.44 0.72 1.6 6 1.03 1.92 1.9 7 0.47 1.35 2.98 0.19 0.33 1.7 9 1.07 0.90 0.8 10  0.33 0.88 2.7 Average 0.93 2.13 2.3CATHEPSIN B ACTIVITY IN INVASIVE HUMAN BREAST CARCINOMA 1 0.63 3.02 4.82 0.51 10.08 19.8 3 0.61 4.43 7.3 4 2.21 2.38 1.1 5 2.06 3.72 1.8Average 1.20 4.73 6.9

[0120] As can be seen from Table 1, all five breast tumors and nine ofthe ten colon tumors showed increased activity of cathepsin B ascompared to matched numbers of normal cells from the same patient (Table1). Increased activity in the colon tumors ranged from 19% to 283%, withan average increase in tumors of greater than two fold. The increase ofcathepsin 2 activity was more pronounced in breast tumors with anaverage increase of slightly less than seven fold.

EXAMPLE 2

[0121] In this example, polymerase chain reaction (PCR) analysis wasperformed. On the basis of previously reported cDNA sequences of 72 kDatype IV collagenase, sense and antisense oligonucleotide primers weresynthesized for amplification of the enzyme activation site (M. Onistoet al, “Reverse Transcription-Polymerase Chain Reaction Phenotyping ofMetalloproteinases and Inhibitors in Tumor Matrix Invasion”, Diagn. Mol.Pathol, 2(2):74-80, 1993). The paired oligonucleotide sequences were:5′-CAA TAC CTG AAC ACC TTC TA, 3′-CTG TAT GTG ATC TGG TTC TTG. LabeledPCR for Single Strand Conformation Polymorphism (SSCP) was obtained bycombining the following in a 10 microliter reaction: 1 microliter 10×PCRbuffer (100 mM Tris-HCL, pH 8.3; 500 mM KCl; 15 mM MgCl₂; 0.1% w/vgelatin); 1 microliter of DNA extraction buffer; 50 pmol of each primer;20 nmol each of dCTP, dGTP, dTTT, and dATP; 0.2 microliter [³²P] dCTP(6000 Ci/mmol); and 0.1 unit Taq DNA polymerase. The amplificationreaction was carried out for 30 cycles at 95° C. for 30 s, 60° C. for 30s, and 72° C. for 30 s.

[0122]FIG. 6a shows the expression of MMP-2 in ten invasive coloncarcinoma cases as compared to normal colonic mucosa from the samepatients. The bar graphs show increases of approximately three fold inthe 72 kDa pro-form of the enzyme (p<0.001) and ten fold in the 62 kDaactive form of the enzyme (p<0.001).

[0123]FIG. 6b shows the expression of MMP-2 in five cases of invasivebreast carcinoma. The bar graphs show an appropriate increase of threefold in the 72 kDa pro-form of the enzyme (p<0.05) and ten fold in the62 kDa active form of the enzyme (p<0.05).

[0124] The 72 kDa pro-type IV collagenase and 62 kDa active form of theenzyme were increased in all ten colon tumors and all five breast tumorsas compared to normal tissue from the same patient. The increase wasgreater in the 62 kDa active form of the enzyme which was elevated anaverage of ten-fold in both the colon and breast tumors as compared tonormal control tissue. The 72 kDa pro-enzyme levels were increased anaverage of three fold in both tumor types. For both breast and colontumors the increase in the 62 kDa active enzyme was more variable thanthat of the pro-enzyme. Elevations in the 62 kDa active enzyme in tumorsranged from 3 to 20 fold while increases in the 72 kDa pro-enzyme wereconsistently in the 2 to 5 fold range. These results are similar to therecent findings of Davis et al (“Activity of Type IV Collagenases inBenign and Malignant Breast Disease”, Br. J. Cancer, 67:1126-1131, 1993)in their analysis of human breast tumors. These authors performedzymogram analysis of tissue sections from human breast cancer patients.These analyses demonstrated that the fraction of total MMP-2 present asthe 62 kDa activated form was statistically elevated in malignantdisease, and a high proportion of this active enzyme species wasdetected in higher grade tumors. The present invention extends thisanalysis by comparing and quantitating both 72 kDa and 62 kDa forms ofthe enzyme in specific regions of invasive tumor and matched normalcontrol epithelium from the same patient.

EXAMPLE 3

[0125] In this example, strand conformation polymorphism (SSCP) analysiswas preformed. Labeled amplified DNA was mixed with an equal volume offormamide loading dye (95% formamide; 20 mM EDTA; 0.05% bromophenolblue, and 0.05% xylene cyanol). The samples were denatured for 5 min at95° C. and loaded onto a gel consisting of 6% acrylamide (49:1acrylamide:bis), 5% glycerol, and 0.6×TBE. Samples were electrophoresedat 8 W at room temperature overnight. Gels were transferred to 3 mmWhatman paper, dried and autoradiography was performed with Kodak X-OMATfilm.

[0126]FIG. 7 shows SSCP analysis of MMP-2 activation site. The figureshows representative cases of normal colon is mucosa compared toinvasive colon carcinoma, and normal breast tissue compared to invasivebreast carcinoma. No difference is observed between the normal and tumorspecimens. The two band in each lane represent single and double formsof DNA. Similar results were obtained for ten colon carcinomas and fourbreast carcinomas.

[0127] To assess if increased tumor levels of activated MMP-2 are due toa mutation in the enzyme, PCR was used to amplify DNA sequence codingfor the activation site of gelatinase A from the colon and breasttumors. The activation site is located 10 kDa from the N-terminus of theenzyme and contains the site of cleavage which converts the 72 kDapro-enzyme into the 62 kDa active species. Amplification and analysis ofthis region by PCR and SSCP showed no detectable mutations in any of theten colon tumors or four breast tumors studied. These results suggestthat increased levels of active enzyme in invasive tumors is most likelydue to a tumor associated activating species. The sensitivity of PCRamplification of DNA from microdissected frozen tissue sections wasdetermined to be less that one high power field. Similar to theamplification of DNA, amplification of mRNA from small cell populationswas preformed according to the present invention using reverse PCR.

[0128] A previous study indicated that MMP-2 is up-regulated in humancolon carcinoma. However, recently several studies using in situhybridization analysis report that the MRNA level of MMP-2 in humancolon carcinoma is increased in the stromal cells as opposed to thetumor cells. In order to address this possibility frozen tissue sectionswere microdissected to measure enzyme levels of MMP-2 in separate tumorand stromal cell populations. From a single high power field sufficienttissue was recovered to quantitate enzyme levels by zymography. Studiesof invasive tumor cells and adjacent stroma from three cases indicatethat 72 kDa pro-MMP-2 and active 62 kDa form are associated with bothtumor cell and stromal cell populations. Preliminary data suggest thatthe highest enzyme levels are at the tumor-stroma interface.

EXAMPLE 4

[0129] Human prostate cancer has been proposed to progress through an insitu tumor phase called prostatic intraepithelial neoplasia (PIN) priorto evolving into overtly invasive cancer. PIN lesions are frequentlyfound in association with prostate carcinoma, and histologically thecells in PIN foci have several features similar to those of invasiveprostate cancer cells. Previous reports have shown that PIN lesions arefrequently aneuploid. However the precise relationship between PIN andinvasive carcinoma has remained unclear.

[0130] In this Example, frozen normal and tumor prostate samples from100 patients treated with transurethral prostatectomy or radialprostatomy were collected. Of these, 30 cases which contained clearlyinvasive cancer as well as at least one focus of identifiable PIN wereselected for study during this Example. Fourteen of the set casescontained more than one focus of PIN. The histopathology of the tumorswas variable and included well-differentiated, moderately differentiatedand poorly differentiated. PIN lesions were both low and high grade.

[0131] Microdissection of selected populations of normal epithelialcells, cells from PIN lesions, and invasive tumor cells from frozentissue sections was performed under direct light microscopicvisualization utilizing the method discussed above and shown in FIGS.8a-8 d. Specific cells of interest were microdissected and procured fromunstained 8 μm frozen sections. In each case, normal epithelium, PINcells, and invasive tumor cells from the same patient were analyzed.

[0132] 5-10 micron sections of formalin-fixed, paraffin-embedded tissueor frozen tissue were prepared on a glass slide according toconventional surgical pathology Protocol. The paraffin sections weredeparaffinized with xylene (×2), 95 ethanol (×2), 50% ethanol (×2),distilled water (×2), and air dried. Frozen or paraffin sections werestained briefly in eosin (1% eosin in 80% ethanol) and air dried.

[0133] An adjacent hematoxylin and eosin section was used to assess thetissue section for optimal areas of microdissection, i.e., localizationof specific small cell populations of interest, exclusion of regionswhich contain significant inflammation, etc.

[0134] Microdissection of selected populations of cells was performedunder direct light microscope visualization. A sterile 30-gauge needlewas used as the transfer surface. Electrostatic interaction between theneedle and cellular material provided the adherence needed to removeselected populations of cells. It was determined that pure cellpopulations of as little as 5 cells could be procured. In addition itwas found possible to procure cells arranged as a single cell layer,i.e., normal epithelium, epithelial lining of cystic lesions, etc.

[0135] Procured cells were immediately resuspended in a 20 ml solutioncontaining 10 mM Tris-HCL, pH 8.0, 100 μm ethylenediamine tetraaceticacid (EDTA), 1% Tween 20, 0.1 mg/ml proteinase K, and incubatedovernight at 37° C. The mixture was boiled for 5 minutes to inactivatethe proteinase K and 0.5-2% of this solution was used for polymerasechain reaction (PCR) analysis.

[0136] The oligonucleotide primers D8S136, D8S137, and NEFL were used tolocate chromosome 8p12-21. Reactions with D8S137 and NEFL were performedin an MJ Research thermal cycler as follows: 2 minutes at 950° C.,followed by 40 cycles of: 950° C. for 30 seconds, 620° C. for 30seconds, 720° C. for 30 seconds, followed by a final 2 minute incubationat 720° C.

[0137] Reactions with D8S136 were cycled as follows: 2 minutes at 950°C., followed by 40 cycles of: 950° C. for 30 seconds, 550° C. for 30seconds, 720° C. for 30 seconds, followed by a final 2 minute incubationat 720° C.

[0138] PCR was performed in 12.5 ml reactions with 200 mM dNTP, 0.8 mMprimers, 2 μl of alpha [³²P] dCTP (NEN), and 1 unit of Taq polymerase.Labeled amplified DNA was fixed with an equal volume of formamideloading dye (95% formamide; 20 mM EDTA; 0.05% bromophenol blue, and 0.05xylene cyanol).

[0139] The samples were denatured for 5 min at 950° C. and loaded into agel consisting of 7% acrylamide (49:1 acrylamide:bis), 5.7 M urea, 32%formamide, and 0.089 M Tris, 0.089 M borate. 0.002 M EDTA (1×TBE).Samples were electrophoresed at 95 Watts for 2-4 hours. Gels weretransferred to 3 mM Whatman paper, and autoradiography was performedwith Kodak X-OMAT film. The criterion for loss of heterozygosity (LOH)was complete, or near complete absence of one allele as determined byvisualization. Cases with LOH showed two alleles in the normalepithelium control and one allele in the tumor or PIN all with similarintensities. Cases with complete or near complete loss (i.e., very faintband) of one allele in tumor or PIN were considered positive for LOH atthat marker.

[0140] The present inventive method was used to microdissect cells fromtissue sections to study loss of heterozygosity on chromosome 8p12-21 inpatients with both prostatic carcinoma and adjacent foci of PIN. Tissuemicrodissection was conducted on 30 patients with concomitant PIN andinvasive prostate cancer. In each case normal epithelium, invasiveprostate cancer and at least one focus of PIN from the same patient wereexamined. In 14 cases multiple foci of PIN were examined. In all caseseach individual PIN lesion and corresponding invasive tumor wereselectively microdissected from adjacent stroma, normal epithelium andinflammatory cells. Essentially pure populations of cells of interestwere procured.

[0141] LOH on chromosome 8p12-21 occurred in at least one PIN lesion in26 of 29 (89.6%) informative cases. Fourteen of the cases contained morethan one PIN lesion. Eleven of these cases showed different allele losspatterns among the PIN lesions, including lose of opposite alleles. Intotal, 8p12-21 LOH was seen in 63.6i (35/55) of PIN lesions studied.Allelic loss of chromosome 8p12-21 was seen in invasive tumors in 28 of29 (96.5%) patients. In contrast with the success associated with theadhesive transfer technique of the present invention, the use of ascraping dissection technique produced an LOH of less than 15%. Thisindicates the sensitivity of the adhesive transfer of the presentinvention is much greater than conventional techniques.

EXAMPLE 5

[0142] Nascent in situ breast carcinomas are frequently observed arisingin association with a spectrum of epithelial hyperplasia and invasivecarcinoma. Pathologists have historically interpreted the commonassociation of atypical hyperplasia, in situ carcinoma and invasivecarcinomas as evidence for a relationship among the entities.

[0143] The polymorphic DNA marker used in this Example was PYGM locatedon chromosome 11q13. Reactions were cycled in a thermal cycler asfollows: 94° C. for 1.5 min., 55° C. for 1 min., 72° C. for 1 min. for atotal of 35 cycles. PCR was performed in 10 μl volumes and contained 1μl 10×PCR buffer (100 mM Tris-Hcl, pH 8.3; 500 mM KCl; 15 mM MgCl₂; 0.1%w/v gelatin; 2 μl of DNA extraction buffer, 50 pM of each primer; 20 nMeach of dCTP, dGTP, dTTP, and dATP; 0.2 μl [³²P]dCTP (6000 Cl/mM); and0.1 unit Taq DNA polymerase. Labeled amplified DNA was mixed with anequal volume of formamide loading dye (95% formamide; 20 mM EDTA; 0.05%bromophenol blue; and 0.05% xylene cyanol). The samples were denaturedfor 5 min. at 95° C. and loaded into a gel consisting of 6% acrylamide(49:1 acrylamide:bis). Samples were electrophoresed at 1800 volts for2-4 hours. Gels were transferred to 3 mM Whatman paper, dried andautoradiography was performed with Kodak X-OMAT film. The criterion forLOH from the microdisseeted in situ and invasive breast samples wascomplete absence of an allele.

[0144] Using the adhesive transfer technique of the present invention,cells were microdissected from normal epithelium, in situ carcinoma andinvasive carcinoma from 8 μm thick formalin fixed deparaffinizedsections from individual biopsies. Allelic loss of chromosome 11q13 wasfound in 69% of human breast carcinoma cases studied (n=105). Theallelic loss was observed in both the in situ and invasive components ofthe tumors. In all cases (26/28) where in situ and invasive cancer waspresent in the same section, the identical allele was lost in the insitu and the invasive carcinoma. This provides molecular support for thelong-held hypothesis that in situ breast cancer is a precursor toinvasive cancer.

[0145] In order to finely map the LOH locus on chromosome 11q13, GenomeCenter provided a series of SSCP probes mapped to the relevant region ofchromosome 11. The initial LOH area was determined to be bracketed bythe proximal marker PYGM, and by the distal marker INT-2. A subset of 20of the 105 cases exhibited LOH of either INT-2 or PYGM, but not both.Using these special cases, a series of intervening markers were used tomap the smallest overlapping region between INT-2 and PYGM which showsLOH. It has been possible to pinpoint the LOH zone to a regionencompassed by only one or two YAG or Cosmid clones at a location whichoverlaps with the MEN-1 (Multiple Endocrine Neoplasia type 1) locus.

[0146] Sequencing gel analysis was performed on PCR-amplified DNAisolated from human tissue microdissected by the method depicted inFIGS. 8a-8 b. The results indicated that microdissection of frozentissue sections allows for more specific analysis of cell populationswithin human tumors than conventional techniques. The microdissectiontechnique of the present invention may be used in combination with anumber of different technologies that allow for analysis of enzymes,mRNA and DNA from pure populations or subpopulations of particular celltypes. This simple technique may have utility in characterizing proteasedistribution during human tumor invasion, precisely determining proteaseexpression in tumor and/or stromal cell populations as an indicator oftumor aggressiveness, and monitoring the effectiveness of anti-proteasetherapeutic agents in inhibiting protease activity at the tumor-stromalinterface. In addition, combination of this microdissection techniquewith PCR, RT-PCR, differential display and SSCP may identify geneticalterations in specific subpopulations of tumor or stromal cell thatwould not be evident in heterogeneous human tumor samples.

EXAMPLE 6

[0147] Standard 6 μm sections from formalin or alcohol-fixed,paraffin-embedded archival tissue samples were prepared on noncoatedglass slides. Sections were deparaffinized, stained with hematoxylin andeosin, treated with 3% glycerol in water for 1 minute and air driedprior to Laser Capture Microdissection (LCM). Fresh tissue, when used,was snap frozen immediately after surgery at −70° C. 6 μm cryostatsections were prepared on standard glass on standard glass histologyslides. Tissue sections were fixed in formalin or alcohol, and stainedwith hematoxylin and eosin (Lerner Laboratories, Pittsburgh, Pa.).Sections were dehydrated in graded alcohols and air-dried for 5 minutesprior to LCM transfer.

[0148] For LCM transfer, 100 μm thick, flat films were made by spreadinga molten ethylene vinyl acetate EVA, (Adhesive Technologies, Hampton,N.J.) onto smooth siliconized or polytetrafluoroethylene surfaces. Theoptically transparent, thin films were placed on top of tissue sections,and the tissue/film sandwich was viewed in an inverted microscope(Olympus Model CK2, Tokyo) at 100× magnification (10× objective). Apulsed carbon dioxide laser beam was introduced via a smallfront-surface mirror coaxial with the condenser optical path so as toirradiate the upper surface of the EVA film. The carbon dioxide laser(either Apollo Company Model 580, Los Angeles, Calif. or CaliforniaLaser Company Model LS150, San Marcos, Calif.) provided the individualpulses of adjustable pulse length and power. A ZnSe lens focused thelaser beam to an adjustable spot size onto the target specimen. For 150mm diameter transfer spots, 25 to 30 mW was delivered to the film duringa 600 msec pulse. For smaller or larger spots, the power was decreasedor increased approximately in proportion to the diameter of the laserspot focused on the target area. The absorption coefficient of the EVAfilm measured both by FT-IR spectrometry and direct transmission wasabout 200 cm⁻¹ at a laser wavelength of 10.6 μm. Since greater than 90%of the laser energy was absorbed within the thermoplastic film, littledirect heating of the tissue specimen occurred. The glass slide provideda large heat sink which served to confine the full-thickness, transient,focally molten plastic with the targeted tissue. After cooling andrecrystallization, the film formed a local surface bond to the targetedtissue stronger than the adhesion forces of the tissue to the slide. Thefilm and targeted cells were removed from the tissue specimen, resultingin focal microtransfer of the targeted tissue to the film surface.

[0149] For polymerase chain reaction, the tissue film and adherent cellswere immediately resuspended in 40 μl of a solution containing 10 mMTris-HCl (pH 8.0), 1 mM EDTA, 1k Tween 20, and 0.1 mg/ml proteinase K,and incubated overnight at 37° C. The mixture was then boiled for 10minutes to inactivate the proteinase K. The tubes were briefly spun(1000 rpm, 1 minute) to remove the film, and 0.5 μl of the supernatantwas used for PCR. For the most efficient transfer recovery, the transferfilm is initially applied to the tissue section as a circular disk ofabout 0.5 cm diameter. After LCM transfer the disk placed into a well ina 96 well microtitre plate containing 40 μl extraction buffer.

[0150] For polymorphic DNA studies, oligonucleotides for the loci D8S136and D8S33 located on chromosome 8q, D17S855 located on chromosome 17q21,D11S449 located on chromosome 11q13, D9S171 on chromosome 9p, specificprimers for exon 2 of the VHL gene, and specific primers forMycobacterium tuberculosis were used (Research Genetics, Huntsville,Ala.). All PCR reactions included incorporation of ³²P-DCTP forvisualization of the PCR product, with the exception of theamplification of M. tuberculosis which was visualized by ethidiumbromide staining.

[0151] For reverse transcription-polymerase chain reaction (RT-PCR),total RNA was extracted from the tissue sample after LCM using amodification of a published RNA microisolation protocol (Stratagene, LaJolla Calif.). Volumes were proportionally adjusted downwards, and a10-fold increase in glycogen carrier (10 ng/ml) was used in allprecipitation steps. After initial recovery and resuspension of the RNApellet, a Dnase step was performed for 3 hours at 37° C. using 10 u/mlof Dnase (GenHunter, Nashville), in the presence of 4 units of Rnaseinhibitor (Perkin Elmer), followed by re-extraction of the RNA. Theintegrity of the RNA sample may be determined by RT-PCR of, for example,actin mRNA using actin-specific primers (Clonetech, Palo Alto, Calif.).The resuspended RNA was reverse transcribed using 5 μM random hexamerprimers (Perkin Elmer), 250 mM dNTPs and 100 units reverse transcriptase(MMLV, GenHunter, Nashville, Tenn.).

[0152] Reverse transcription was accomplished by heating the RT mix(without enzyme) to about 65° C. for 5 minutes, followed by primerannealing for 10 minutes at about 25° C. The reverse transcriptase wasthen added, followed by further incubation at 25° C. for 10 minutes, 37°C. for 40 minutes and 94° C. for 5 minutes. PCR was performed withspecific actin or PSA primers, and the products subjected to denaturingelectrophoresis gel analysis.

EXAMPLE 7

[0153] cDNA libraries were generated using material isolated by lasercapture microdissection. Double stranded cDNA is prepared fromapproximately 5 μg of total cellular RNA based on the RNaseH-mediatedsecond strand replacement method. The reverse transcription first strandsynthesis was primed with about 50 ng/μl of oligo(dT). The reaction wascarried out at about 45° C. for 15 minutes. The second strandreplacement and addition of EcoRI linkers were performed as suggested bythe manufacturer (Superscript Choice System, Life Technologies Inc.,Gaithersburg, Md.). After second strand synthesis, the reaction productwas electrophoresed and fragments from about 0.3 kb to about 2 kb weregel isolated from 1% low melting point agarose using beta-agarose (NewEngland Biolabs, Beverly, Mass.). The cDNA pellet was resuspended in 20μl Tris-EDTA and stored at −20° C.

[0154] Five microliters of the isolated cDNA was amplified by 5 cyclesof PCR under standard conditions and the linker-specific primer LINKthat also functions to direct UDG cloning. The cDNA was first denaturedfor 3 minutes at 95° C., followed five time by the following cycle: 15seconds at 95° C.; 15 seconds at 55° C.; and 2 minutes at 72° C. A finalextension was performed for 5 minutes at 72° C. The oligonucleotideprimers, nucleotides, enzyme, etc. were removed from the reaction mix bycolumn chromatography (CHROMA SPIN-200, Clontech, Palo Alto, Calif.).The column flow through was ethanol precipitated and resuspended in 20μl of Tris-EDTA. Six microliters of the product was cloned into the UDGcloning vector pAMP10 according to the manufacturer's instructions (LifeTechnologies Inc., Gaithersburg, Md.). A complex and relatively lowredundancy library of approximately 200,000 clones was prepared in thisway.

[0155] cDNA libraries produced under the practice of the invention havemany advantages not heretofore attainable, such as being derived from ahomogeneous cell type, whether normal or abnormal, as opposed to beingfrom heterogeneous tissue or transformed tissue culture cell lines.Moreover, the cDNAs libraries permit the comparative analysis of mRNAexpression during development, aging, neoplastic transformation, etc.The cDNA libraries of the invention, thus, are useful for measuring thesimultaneous fluctuations of expression of multiple genes or geneticalterations occurring in developing or diseased tissues.

[0156] The present invention has applications in routine diagnosis ofhuman tumors including microdissection of pre-malignant lesions of alltypes of cancer, genetic analysis of infectious diseases, gene therapy,tissue transformation, and gene localization and analysis of transgenicanimals. Additional applications of this technique include analysis ofthe genotype, cellular products, or infesting organisms of rarepopulations such as monocytes infected with drug resistant organisms,Reed-Sternberg cells of Hodgkins disease, Kaposi's sarcoma cells, stemcells, and vessel cells. Moreover, genetic analysis, or identificationof, micro-organisms infesting microscopically visualized cells intissues, lymph nodes or inflammatory areas can also be accomplished withhigh precision.

[0157] Although the present invention has been described with referenceto particular means, materials and embodiments, one skilled in the artcan easily ascertain the essential characteristics of the presentinvention and various changes, modifications and alterations may be madeto adapt the various uses and characteristics without departing from thespirit and broad scope of the present invention as described by theclaims which follow.

What is claimed is:
 1. An activatable film for activated use in capturemicrodissection where a source emits electromagnetic energy outside of arange of human vision, the activatable film comprising: a film having anormal non-adherence to a biological sample; the film opticallytransparent in the range of human vision for permitting the biologicalsample to be viewed through the film; the film activatable upon heatingfor becoming adhesive at an activated region for adhering to abiological sample at the activated region; and, a dye on the film whichis optically transparent, the dye coupling to and transducing theelectromagnetic energy outside of the range of human vision to heat andactivate the film to become adhesive at the activated region.
 2. Anactivatable film for activated use in capture microdissection accordingto claim 1 and wherein: the film includes a region for indicia.
 3. Anactivatable film for activated use in capture microdissection accordingto claim 1 and wherein: the film is coated with a release agent to avoidbonding to a biological sample.
 4. An activatable film for activated usein capture microdissection according to claim 1 and wherein: the filmincludes a support substrate; and, an activation substrate.
 5. Anactivatable film for activated use in capture microdissection accordingto claim 4 and wherein: the activation substrate is chosen from a groupconsisting of an emulsion layer, a coated film, a separate impregnatedweb, thermal sensitive adhesives and waxes, hot glues and sealants,ultraviolet sensitive adhesives, ultraviolet sensitive waxes,ultraviolet sensitive curing optical adhesives, and thermal emulsions.6. An activatable film for activated use in capture microdissectionaccording to claim 1 and wherein: the transfer film can be made from afilm selected from the group consisting of ethylene vinyl acetate (EVA),polyurethanes, polyvinyl acetates, thermal sensitive adhesives, thermalsensitive waxes, thermally-activated hot glues, thermally-activatedsealants, ultraviolet sensitive curing optical adhesives, thermalemulsions, high mesh powdered reconstituted lelt fixit emulsion, acetal,acrylic, alloys, allyl, bismaleimides, cellulosics, epoxy,fluoroplastics, ketone-based resins, liquid crystal polymers,melamine-formaldehyde, nitrile, nylon, phenolic, polyamide,polyacrylate, polybenzimidazole, polybutylene, polycarbonate,thermoplastic polyester, liquid crystal polymers, polybutyleneterephthalate (PBT), polycyclohexvlenedimethylene terephthalate (PCT),engineering grade polyethylene terephthalate (PET), standard gradepolyethylene terephthalate (PET), thermoset polyetherimide polyethylenepolyester, branched polyethylene, ethylene acid copolymer,ethylene-ethyl acrylate (EEA), ethylene-methyl acrylate (EMAC),ethylene-vinyl alcohol copolymers (EVOH), high-density polyethylene,HMW-high-density polyethylene, Ionomer, linear low-density polyethylene,linear polyethylene, low-density polyethylene, UHMW polyethylene, verylow-density polyethylene, thermoplastic polyimide, thermoset polyimide,polymethylpentene, modified Polyphenylene oxide, polyphenylene sulfide,blow molding PPS, polyphthalamide, polypropylene, polypropylenehomopolymer, polypropylene impact copolymers, polypropylene randomcopolymers, silicones styrenic resins, ABS, ACS,acrylic-styrene-acrylonitrile, expandable polystyrene, general purposepolystyrene, impact polystyrene, olefin-modified SAN, polystyrene,styrene-acrylonitrile (SAN) and styrene-butadiene copolymers.
 7. Anactivatable film for activated use in capture microdissection accordingto claim 4 and wherein: the support substrate is transparent.
 8. Anactivatable film for activated use in capture microdissection accordingto claim 1 and wherein: the support substrate is chosen from the groupconsisting of a transparent polymer and transparent glass.
 9. Anactivatable film for activated use in capture microdissection accordingto claim 1 and wherein: the dye couples to and transduces energy in aninfrared range.
 10. An activatable film for activated use in capturemicrodissection according to claim 1 and wherein: the dye is selectedfrom a group consisting of pytalocyanine dyes, indocyanine dyes, and,naphthanlocyanine dyes.
 11. An activatable film for activated use incapture microdissection according to claim 1 and further including: thefilm is a thermoplastic film.
 12. An activatable film for activated usein capture microdissection according to claim 1 and further including: amarker added to the film for indicating activation.
 13. In combination:a biological sample; a microscope for viewing the biological sample at aselected portion; a light source for illuminating the biological samplein a range of human vision for view in the microscope; an improvement tothe combination comprising: a film having a normal non-adherence to thebiological sample; the film optically transparent in the range of humanvision for permitting the biological sample to be viewed by themicroscope through the film; the film activatable upon heating forbecoming adhesive at an activated region for adhering to a biologicalsample at the activated region; and, a dye on the film which isoptically transparent, the dye coupling to and transducingelectromagnetic energy outside of the range of human vision to heat andactivate the film to become adhesive at the activated region; and, asource of electromagnetic energy outside of the range of human visionfor being locally directed on the dye on the film overlying the selectedportion of the biological sample to couple to the dye, heat the film,and activate the film to become adhesive for adhering to the selectedportion of the biological sample; means for moving the film intoapposition with biological sample; and, means for directing the sourceof electromagnetic energy to the film in apposition whereby selectedcellular material from the biological sample is adhered to the film. 14.The combination according to claim 13 and further including: the dyecouples to and transduces energy in an infrared range.
 15. Thecombination according to claim 13 and further including: the film is athermoplastic film.
 16. The combination according to claim 13 andfurther including: a marker added to the film for indicating activation.17. A method of direct extraction of cellular material from a tissuesample which comprises: providing a tissue sample; providing a transfersurface which only upon activation at selected regions has a property toprovide the selected regions thereof with adhesive characteristics tothe tissue sample; juxtaposing the tissue sample with the transfersurface; identifying at least one portion of the tissue sample which isto be extracted; activating a region on the transfer surface with pulsedradiation so that the selected region of the transfer surface adheres tothe at least one portion of the tissue sample while preserving focaltissue morphology; separating the transfer surface from the tissuesample while maintaining adhesion between the selected region of thetransfer surface and the at least one portion of the tissue sample sothat the at least on portion of the tissue sample is extracted from aremaining portion of the tissue sample.
 18. A method of directextraction of cellular material from a tissue sample according to claim17 which comprises: observing the tissue sample during the separatingstep.
 19. A method of direct extraction of cellular material from atissue sample which comprises: providing a tissue sample; providing aslide for mounting of the tissue sample having a first adhesiveattraction to the tissue sample; mounting the tissue sample to theslide; providing a transfer surface which only upon activation atselected regions has a property to provide the selected regions thereofwith adhesive characteristics with a second and greater adhesiveattraction to the tissue sample; juxtaposing the tissue sample with thetransfer surface; identifying at least one portion of the tissue samplewhich is to be extracted; activating a region on the transfer surfacewith radiation so that the selected region of the transfer surfaceadheres to the at least one portion of the tissue sample whilepreserving focal tissue morphology; separating the transfer surface fromthe tissue sample while maintaining adhesion between the selected regionof the transfer surface and the at least one portion of the tissuesample so that the at least one portion of the tissue sample isextracted from a remaining portion of the tissue sample.
 20. A method ofdirect extraction of cellular material from a tissue sample according toclaim 19 which comprises: dipping the provided slide in an aqueousglycerol solution; and, drying the slide before the placing of thetissue sample on the slide.
 21. A method of direct extraction ofcellular material from a tissue sample according to claim 19 whichcomprises: providing an enclosing material having a high bondingstrength to the transfer surface; enclosing the tissue sample in theprovided material before placing the tissue sample on the slide.
 22. Amethod of direct extraction of cellular material from a tissue samplewhich comprises: providing a tissue sample; providing a transfer surfacewhich only upon activation at selected regions has a property to providethe selected regions thereof with adhesive characteristics to the tissuesample, the transfer surface including material chosen from a groupconsisting of thermoplastic materials, polymerizable substances, thermalselective adhesives and waxes, thermally activated hot glues andsealants, ultraviolet sensitive and curing optical adhesives, andthermal and optical emulsions; juxtaposing the tissue sample with thetransfer surface; identifying at least one portion of the tissue samplewhich is to be extracted; activating a region on the transfer surfacewith laser radiation so that the selected region of the transfer surfaceadheres to the at least one portion of the tissue sample whilepreserving focal tissue morphology; separating the transfer surface fromthe tissue sample while maintaining adhesion between the selected regionof the transfer surface and the at least one portion of the tissuesample so that the at least on portion of the tissue sample is extractedfrom a remaining portion of the tissue sample.
 23. A method of directextraction of cellular material from a tissue sample according to claim22 which comprises: the provided transfer surface includes a supportsubstrate substantially transparent at a visible wavelength and anactivation wave length; and, an activation substrate for activation atthe activation wave length.
 24. A method of direct extraction ofmaterial from a sample which comprises: providing a sample; providing atransfer surface which only upon activation at selected regions has aproperty to provide the selected regions thereof with adhesivecharacteristics to the sample; juxtaposing the sample with the transfersurface; identifying at least one portion of the sample which is to beextracted; activating a region on the transfer surface with radiation sothat the selected region of the transfer surface adheres to the at leastone portion of the sample; separating the transfer surface from thesample while maintaining adhesion between the selected region of thetransfer surface and the at least one portion of the sample so that theat least one portion of the sample is extracted from a remaining portionof the sample.
 25. A method of direct extraction of material from asample according to claim 24 which comprises: identifying the at leastone portion of the sample from a first direction relative to the sample;and, activating a region on the transfer surface from a second andopposite direction relative to the sample.
 26. A method of directextraction of material from a sample according to claim 24 whichcomprises: identifying the at least one portion of the sample from firstdirection; and, activating a region on the transfer surface withradiation from the first direction.
 27. A method of direct extraction ofmaterial from a sample according to claim 24 which comprises: providinga sample includes providing a non-biological sample.
 28. A method ofdirect extraction of material from a sample according to claim 24 whichcomprises: providing a sample chosen from the group consisting of a cellsection, a microtome section, and a cell smear.
 29. A method of directextraction of material from a sample according to claim F1 whichcomprises: the microtome section is chosen from the group consisting ofparaffin embedded and formalin-fixed tissue samples.
 30. A method ofdirect extraction of material from a sample according to claim 24 andwherein the step of juxtaposing the sample with the transfer surfaceincludes: fixing the transfer material to the sample.
 31. A method ofdirect extraction of material from a sample according to claim F2wherein the step of fixing the transfer material to the sample is chosenfrom at least one of the following group consisting of clips, guides,tape, and standard adhesives.
 32. A method of direct extraction ofmaterial from a sample according to claim 24 which comprises: providinga transfer surface which is activated by exposure to activationprocessing selected from the group consisting of electromagnetic energy,a heat source, electrically heated radiant probes, flashbulb generatedenergy, and focused xenon lamps.
 33. A method of direct extraction ofmaterial from a sample according to claim 24 which comprises: activatinga region on the transfer surface with radiation so that the activatedregion of the transfer surface adheres to the at least one portion ofthe sample, the radiation produced by a laser chosen from the groupconsisting of CO₂ laser, laser diodes, tuneable frequency lasers,sapphire lasers, diode-pumped NdYAG lasers, and lasers having tunableoutput from infrared to ultraviolet.